Is S.O.C. medium absolutely required when recovering competent bacterial cells during transformation?

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Many media can be used to grow transformed cells, including standard LB, SOB or TB broths. However, S.O.C. is the optimal choice for recovery of the cells before plating. The nutrient-rich formula with added glucose is often important for obtaining maximum transformation efficiencies.

Answer Id: E3100

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What strain should I use to transform my library?

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OmniMAX™ 2 is the preferred strain for transforming libraries because of its high transformation efficiency and genomic cloning compatibility characteristics.

Answer Id: E6699

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Can encapsulated phagemid DNA or M13 phage be used to infect bacteria?

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Answer

Single-stranded DNA viral particles like M13 require the presence of an F pilus in order to infect E. coli. This criterion is met by TOP10F', DH5α F'IQ™, INVαF', Stbl4™, OmniMAX™2-T1 and DH12S™ cells. These cells are not traD mutants, which effectively allows the cells to retain the F' episome. Transforming single-stranded DNA can cause a 100- to 1,000-fold reduction in efficiency compared to viral particles.

Answer Id: E3101

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How can AmpliTaq™ DNA Polymerase be inactivated after PCR?

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Answer

There are several approaches that can be taken to inactivate the AmpliTaq™ DNA Polymerase after PCR.

(1) Because AmpliTaq™ DNA Polymerase is thermostable, it is necessary to heat it to high temperatures in order for it to be inactivated. Typically, a 99-100 degrees C for 10 min is sufficient.

(2) Raising the EDTA concentration to 10 mM will chelate any free Mg2+. Mg2+ is necessary for enzyme activity. By removing the Mg2+ the enzyme will no longer exhibit enzyme activity.

(3) Phenol-chloroform extraction of the PCR product and ethanol precipitation will also inactivate AmpliTaq™ DNA Polymerase.

Answer Id: E1319

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What advantages do your Stbl2™ cells offer over other cloning strains?

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Answer

There are other strains available that may function similarly to Stbl2™ cells in stabilizing inserts or vectors with repeated DNA sequences. However, one advantage of Stbl2™ cells over many similar strains is that they are sensitive to Kanamycin, so you can use Stbl2 to propagate plasmids containing a Kanamycin resistance marker. 

Answer Id: E4289

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Does the methylation status of DNA affect its ability to be cloned?

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Yes. Bacterial host cells will often degrade incoming DNA that has a methylation pattern that is "foreign" relative to that of the cell. Several host strains have been modified to accept mammalian methylation patterns. The modified markers include mcrA, mcrBC, and mrr. Also, endogenous (b-type) restriction endonucleases can be problematic. Modifications of the host to be rK- or rB- are necessary and include hsdR17(AK-, MK+), hsdR17(rK-, mK-), hsdS20(rB-, rB-) or hsdRMS. Strains with the hsdR17(rK-, mK+) mutation lack K-type restriction endonuclease, but contain K-type methylase. DNA prepared from hosts that are rK- mK- is unmethylated and will transform with lower efficiency in rK+ hosts.

TOP10, DH10B™, and OmniMAX™2-T1 cells contain the mcr, mrr, and hsdRMS mutations. Mach1 and standard DH5α™ strains only have the hsdR17(rK- mK+) mutation and are not recommended for cloning eukaryotic genomic DNA.

Answer Id: E3102

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When should DMSO, formamide, glycerol and other cosolvents be used in PCR?

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Cosolvents may be used when there is a failure of amplification, either because the template contains stable hairpin-loops or the region of amplification is GC-rich. Keep in mind that all of these cosolvents have the effect of lowering enzyme activity, which will decrease amplification yield. For more information see P Landre et al (1995). The use of co-solvents to enhance amplification by the polymerase chain reaction. In: PCR Strategies, edited by MA Innis, DH Gelfand, JJ Sninsky. Academic Press, San Diego, CA, pp. 3-16.

Additionally, when amplifying very long PCR fragments (greater than 5 kb) the use of cosolvents is often recommended to help compensate for the increased melting temperature of these fragments.

Answer Id: E1320

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Do any Invitrogen™ competent cells contain DMSO in the freezing medium?

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Answer

Yes, several of our competent cells products are frozen with DMSO. The presence of DMSO (dimethylsulfoxide) will generally be indicated in the MSDS files if you have a question about a particular product, but here is a list of commonly used products that are known to have DMSO in the freezing buffer:

One Shot™ OmniMAX™ 2 T1 Phage Resistant Cells, Cat. No. C8540-03

One Shot™ INVαF' Chemically Competent Cells, Cat. No. C2020-03 and C2020-06

One Shot™ MAX Efficiency™ DH5α-T1™ Chemically Competent Cells, Cat. No. 12297-016

MAX Efficiency™ DH5α-T1™ Phage Resistant Cells, Cat. No. 12034-013

MAX Efficiency™ DH5α™ Chemically Competent Cells, Cat. No. 18258-012

Library Efficiency™ DH5α™ Chemically Competent Cells, Cat. No. 18263-012

MAX Efficiency™ DH5α F'IQ™ Cells, Cat. No. 18288-019

MAX Efficiency™ Stbl2™Chemically Competent Cells, Cat. No. 10268-019

Answer Id: E3355

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How do you recommend that I prepare my DNA for successful electroporation of E. coli?

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Answer

For best results, DNA used in electroporation must have a very low ionic strength and a high resistance. A high-salt DNA sample may be purified by either ethanol precipitation or dialysis.

The following suggested protocols are for ligation reactions of 20ul. The volumes may be adjusted to suit the amount being prepared.

Purifying DNA by Precipitation: Add 5 to 10 ug of tRNA to a 20ul ligation reaction. Adjust the solution to 2.5 M in ammonium acetate using a 7.5 M ammonium acetate stock solution. Mix well. Add two volumes of 100 % ethanol. Centrifuge at 12,000 x g for 15 min at 4C. Remove the supernatant with a micropipet. Wash the pellet with 60ul of 70% ethanol. Centrifuge at 12,000 x g for 15 min at room temperature. Remove the supernatant with a micropipet. Air dry the pellet. Resuspend the DNA in 0.5X TE buffer [5 mM Tris-HCl, 0.5 mM EDTA (pH 7.5)] to a concentration of 10 ng/ul of DNA. Use 1 ul per transformation of 20 ul of cell suspension.

Purifying DNA by Microdialysis: Float a Millipore filter, type VS 0.025 um, on a pool of 0.5X TE buffer (or 10% glycerol) in a small plastic container. Place 20ul of the DNA solution as a drop on top of the filter. Incubate at room temperature for several hours. Withdraw the DNA drop from the filter and place it in a polypropylene microcentrifuge tube. Use 1ul of this DNA for each electrotransformation reaction.

Answer Id: E4159

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Why is it necessary to dilute ligated DNA products before adding them to competent bacterial cells?

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Answer

Components of the ligation reaction (enzymes, salts) can interfere with transformation, and may reduce the number of recombinant colonies or plaques. We recommend a five-fold dilution of the ligation mix, and adding not more than 1/10 of the diluted volume to the cells. For best results, the volume added should also not exceed 10% of the volume of the competent cells that you are using.

Answer Id: E3098

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